Transepithelial transport of iodide in thyroid involves at least two pathways, one the transport of iodide from the blood across the basal membrane into the thyroid follicular cell where iodide is concentrated 20- 100 fold; and a second associated with iodide loss from the cell across the apical membrane into the follicular lumen where thyroglobulin is iodinated and thyroid hormones formed. Goals of this project include the characterization, identification, and isolation of these "iodide transport proteins". To aid in these studies, we have used chloride channel blockers as potential probes of iodide transport, and have used a cultured rat thyroid cell line (FRTL-5 cells) as these cells have all the properties of iodide transport described in intact thyroid tissue. We found that stilbene derivatives enhance iodide uptake, and that the anion exchange protein AE2 is expressed in high levels in these cells, but the expression is not regulated by TSH. Studies in FRTL-5 cells suggest that the anion exchanger may play a role in iodide efflux in these cells. In contrast derivatives of N-substituted anthranilic acid were found to depress iodide entry into these cells. These compounds block chloride channels in a variety of epithelial cell types. One compound competitively inhibited sodium iodide uptake in FRTL-5 cells and in plasma membrane vesicles made from pig thyroid. A derivative of this compound, 2-(4- aminocyclooctylamino)-5-nitro-benzoic acid, was prepared to use as a photo-affinity probe. Another approach to identify the NaI transport protein has been expression cloning that uses a cDNA expression library, prepared from cultured rat thyroid cells superinduced to actively transport iodide, and a replica plate assay for screening. These studies are still in progress.